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Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. [ABSTRACT FROM AUTHOR]

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane — that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex. [ABSTRACT FROM AUTHOR]

Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 Å resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded β-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-α receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-α-mediated NF-κB activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity. [ABSTRACT FROM AUTHOR]

Amyloid precursor protein (APP) plays a central role in Alzheimer disease. A proteolytic-breakdown product of APP, called β-amyloid, is a major component of the diffuse and fibrillar deposits found in Alzheimer diseased brains. The normal physiological role of APP remains largely unknown despite much work. A knowledge of its function will not only provide insights into the genesis of the disease but may also prove vital in the development of an effective therapy. Here we describe the 1.8 Å resolution crystal structure of the N-terminal, heparin-binding domain of APP (residues 28?123), which is responsible, among other things, for stimulation of neurite outgrowth. The structure reveals a highly charged basic surface that may interact with glycosaminoglycans in the brain and an abutting hydrophobic surface that is proposed to play an important functional role such as dimerization or ligand binding. Structural similarities with cysteine-rich growth factors, taken together with its known growth-promoting properties, suggests the APP N-terminal domain could function as a growth factor in vivo. [ABSTRACT FROM AUTHOR]